Background

In rheumatoid arthritis (RA), constitutive activation of integrins promotes retention
of oxidatively stressed, activated T lymphocytes in synovial tissue where they perpetuate
inflammation through cell–cell contact with synovial stromal cells, macrophages and
plasma cells. We have previously demonstrated that activation of Ras GTPases, coupled
with inactivation of the related GTPase Rap1, is responsible for oxidative stress
in RA synovial fluid (SF) T lymphocytes [1]. Inactivation of Rap1, however, is incongruous with the observation that Rap1 signaling
is absolutely required for integrin function in T lymphocytes. Because T lymphocytes
express multiple Ras homologs (H-Ras, K-Ras, and N-Ras), we explored the possibility
that these proteins may make distinct contributions to reactive oxygen species (ROS)
regulation and integrin function in T lymphocytes.

Objectives

To determine which Ras homologs are activated in RA SF T lymphocytes, to determine
whether Ras homologs selectively regulate ROS production and integrin-dependent adhesion
in T lymphocytes, and to use pharmacological inhibitors to characterize downstream
signaling pathways by which Ras homologs may mediate these effects.

Results

All three homologs of Ras were found to be constitutively activated in RA SF T lymphocytes.
Active H-Ras, but not K-Ras or N-Ras, stimulated ROS production in both Jurkat and
peripheral blood T lymphocytes. H-Ras-induced ROS production was sensitive to BAPTA-AM
and catalase, but not LY294002 or DPI, indicating a calcium-sensitive hydrogen peroxide
source of ROS that does not require a functional NADPH oxidase. These results are
consistent with our previous observations in purified RA SF T lymphocytes. In contrast,
N-Ras, but not H-Ras or K-Ras, induced integrin-dependent adhesion in Jurkat T cells
and peripheral blood T lymphocytes. N-Ras-induced adhesion was sensitive to LY294002,
indicative of PI3-kinase signaling. Additionally, inactivation of Rap1 with RapGAP
had no effect on N-Ras-induced adhesion. This represents the first (patho)physiological
signaling pathway by which integrins can be activated independently of Rap1. Our results
suggest that selective targeting of H-Ras and N-Ras may allow specific modulation
of T-cell ROS production and integrin activation in RA T lymphocytes.